High-throughput screening kinetic analysis structural study and biochemical characterization of a high-efficiency fungal laccase DLac

  • 吳 孟璇

Student thesis: Doctoral Thesis

Abstract

Fungal laccase is a blue glycosylated four-copper oxidase that catalyzes the oxidation of phenolic units in lignin as well as a number of phenolic compounds and aromatic amines A high-efficiency laccase DLac was secreted by an indigenous fungal strain Cerrena sp RSD1 isolated from rice straw compost it is identified using 18S rDNA and internal transcribed spacer sequencing analysis A procedure of submerged culture using rice straw as a feedstock was carried out to produce DLac with high catalytic efficiency of 1 5×109 s-1 M-1 show the enzyme to be diffusion-limited So far only a few study focus on structural properties of the diffusion-limited enzyme it is important to investigate the crucial structural features that govern the enzyme kinetics The crystal structure of DLac was determined at 1 38 ? resolution DLac displays the typical fungal laccase architecture consisting of three domains and each domain is folded into the greek key β-barrel topology Four copper atoms were coordinated with His and Cys residues to create the catalytic site The crystal structure was determined to atomic resolution and its overall structure was found to be closely homologous to the monomeric laccases However DLac displays some unique substrate-binding loops different from those in other laccases The substrate-binding residues with small side-chains and the short substrate-binding loop IV widen the substrate-binding cavity and this may facilitate access by larger substrates DLac is not as highly-glycosylated as other fungal laccases and contains one highly-conserved glycosylation site at N432 and another unique site at N468 The N-glycans stabilize the substrate-binding loops and the protein structure and the first N-acetylglucosamine is crucial for catalytic efficiency A submerged culture method useful for industrial application allows a five-fold increase of protein yield to be achieved Laccases that are tolerant to organic solvents are powerful bio-catalysts with broad applications in biotechnology most frequently carried out at high concentration of solvent during which process the proteins can be unfolded and enzyme activity can be lost In this study it will be shown that pre-incubation of fungal laccases with organic solvents can result in an effective (and reversible) 1 5 to 4 0 fold enhancement of enzyme activity Several organic solvents including acetone methanol ethanol DMSO and DMF were effective in this specific enhancement in all the laccases studied The enhancement was not substrate-specific and could be observed in both phenolic and non-phenolic substrates Although laccase pre-incubated with organic solvents was sensitive to high temperature it remained stable at 25°C which was an advantage for long term storage The 3-D structure of DLac pre-incubated with acetone was determined and it was confirmed that the protein structure remained intact and stable at high concentrations of organic solvent Furthermore the turnover rate of fungal laccases was improved by pre-incubation in an organic-solvent and DLac showed the highest enhancement among the fungal laccases examined This investigation has shed light on improving fungal laccase usage under extreme conditions and has extended the opportunities for bioremediation decolorization and organic synthesis
Date of Award2019
Original languageEnglish
SupervisorHao-Jen Huang (Supervisor)

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